US6127340A - Serine protease inhibitors - Google Patents

Serine protease inhibitors Download PDF

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US6127340A
US6127340A US08/894,120 US89412098A US6127340A US 6127340 A US6127340 A US 6127340A US 89412098 A US89412098 A US 89412098A US 6127340 A US6127340 A US 6127340A
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Donovan St. Clair Green
Said Mohammed Anwr Ahmed Elgendy
Geeta Patel
Michael Finbar Scully
Christopher Andrew Goodwin
Vijay Vir Kakkar
John Joseph Deadman
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Trigen Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/75—Fibrinogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
    • C07F5/02—Boron compounds
    • C07F5/025—Boronic and borinic acid compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6564—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms
    • C07F9/6571—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and oxygen atoms as the only ring hetero atoms
    • C07F9/657163—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and oxygen atoms as the only ring hetero atoms the ring phosphorus atom being bound to at least one carbon atom
    • C07F9/657181—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and oxygen atoms as the only ring hetero atoms the ring phosphorus atom being bound to at least one carbon atom the ring phosphorus atom and, at least, one ring oxygen atom being part of a (thio)phosphonic acid derivative
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06191—Dipeptides containing heteroatoms different from O, S, or N
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to enzyme inhibitors and enzyme substrates, particularly those of trypsin-like or chymotrypsin-like enzymes, and to their use for example in the treatment or prevention of thrombosis.
  • Cardiovascular disease is a major cause of mortality, with incidence across the world higher than that of cancer.
  • Acute events in the disease state such as myocardial infarction, stroke, peripheral arterial occlusion and venous thromboembolic disease have recently been understood to be precipitated by formation of thromboembolic clots.
  • This clot formation, as well as the aetiology of the disease state, e.g. formation of atheromatous plaque has been shown to be mediated by the coagulation serine protease enzymes which control also the normal haemostatic balance of the blood. Modulation of any one coagulation protease, especially Factor VIIa. Factor Xa or thrombin, has been shown to control thrombogenesis. This has led to the development of inhibitors of serine protease enzymes to prevent thrombotic events in the clinic.
  • Serine protease inhibitors have been designed which use functional groups, e.g. CO--H, B(OH) 2 , P(O)(OR) 2 , beta lactam, chloromethylketone, to interact with the triad and thereby block activation of the substrates.
  • inhibitors selective for one target protease Such selective inhibitors have been prepared by making peptide inhibitors comprising peptide sequences that bind preferentially to subsites unique in the target protease. Typically these sequences mimic the structure around the scissile bond of the natural substrate of the protease, which is fibrinogen in the case of thrombin.
  • selective peptide inhibitors of thrombin typically incorporate a sequence based on Phe-Pro, or more generally (aa)-Pro, where (aa) is some hydrophobic amino acid or analogue thereof
  • amino acid residue which provides the carbonyl group of the scissile bond of a peptide sequence is designated "P1".
  • P1 are designated P2, P3, P4, . . . etc; amino acid residues on the C-terminal side of residue P1 are designated P1', P2', P3'. . . .
  • P1' is glycine and P2' is proline.
  • the protease contains a "specificity pocket" which recognises the side chain of the P1 amino acid.
  • Thrombin belongs to a family of serine protease inhibitors described as "trypsin-like”: the trypsin-like proteases normally recognise P1 residues with arginine-like or serine-like side chains.
  • trypsin-like the trypsin-like proteases normally recognise P1 residues with arginine-like or serine-like side chains.
  • There is also a chymotrypsin-like family of serine protease inhibitors whose specificity pocket recognises phenylalanine-like and alanine-like side chains on the P1
  • Peptide inhibitors of serine proteases have been made in which the P1 terminal carboxy group is replaced by another acid group, e.t. a boronic acid group or a phosphorus oxyacid function.
  • the P1 terminal carboxy or heteroatom analogue group may be derivatised, for example to form an ester, an alcohol, a thiol or an amine or to replace the OH groups of boronic acid with fluorine.
  • the identity of the derivative moiety is not critical and may be selected according to the desired use of the target compound.
  • Peptide inhibitors having a boron or phosphorus heteroatom analogue group at the P1 residue are described in, for example, WO 92/07869 and EP 0471651.
  • ⁇ -amino boronic acid peptides have been prepared because of the favourable binding energy of the interaction of boron with a nucleophile, such as the lone pair of the Ser hydroxyl or His imidazole group, to give a tetrahedral boronate intermediate which mimics the shape of the "transition state" formed during substrate cleavage and so is tightly bound to the enzyme.
  • the ⁇ -amino group of such ⁇ -amino boronic acid compounds forms the P1-P2 amide link of the peptide.
  • Kettner The studies of Kettner have shown that the strongest interactions, intimated by the best observed inhibition constants, are achieved by "substrate-like" inhibitors where the boron interacts with the active site serine, while “non-substrate-like” inhibitors, where the boron interacts only with the active site histidine, bind more weakly.
  • the binding interactions at the subsites determine the geometry of the active site group, where the amide bond between the P2 and P1 groups confers a rigid "amide plane" geometry on the system, with usually trans orientation of substituents and typically 1.3 ⁇ CO--NH bond lengths.
  • ⁇ -amino group is critical to the activity of peptide inhibitors. and forms a hydrogen bond to the inhibited enzyme, e.g. P1- ⁇ -amino of PPACK, NAPAP or MQPA to Gly-216 of thrombin (Bauer, M., Brandsetter, H.; Turk, D.; Sturzebecher, J. and Bode, W. (1993). Seminars in Thrombosis and Haemostasis, 19, 352-360).
  • P1- ⁇ -amino of PPACK, NAPAP or MQPA to Gly-216 of thrombin
  • EP 0118280 and equivalent U.S. Pat. Nos. 4,638,047 and 4,772,686 describe peptide thrombin inhibitors comprising amino acid residues on the C-terminal side of the scissile bond in which the P1-P1 1 scissile peptide bond is replaced by a non-hydrolysable isosteric linkage, namely --COCH 2 --, --CHOHCH 2 -- or CH 2 NH--.
  • peptide serine protease inhibitors include peptide serine protease inhibitors. It will be understood that the term "peptide” includes peptide analogues. Such inhibitors are known to have at the carboxy position of the P1 residue an optionally derivatised carboxy group or an optionally derivatised heteroatom analogue of a carboxy group.
  • aa 1 , aa 2 , and aa 3 represent natural or unnatural acid residues and (aa 4 ) m one or more optional amino acid residues linked to the amino group of aa 3 .
  • any one or more aa groups may be analogues of amino acid residues in which the ⁇ -hydrogen is replaced by a substituent.
  • X represents H or a substituent on the N-terminal amino group
  • Z is --COOH or a C-terminal extension group (carboxy replacement group), for example as known in the art.
  • Z is a heteroatom acid group, e.g.
  • Preferred heteroatom analogue groups are --B(OH) 2 and --P(O)(OM) 2 ; a less preferred heteroatom analogue group is S(O) 2 OH.
  • Derivatives of the acid groups include those in which inert organic groups, typically containing no more than 20 carbon and hetero-atoms, replace the hydrogen of any acid --OH group; the inert organic groups may be joined to the acid group through the intermediary of a functional group, such as carbonyl or amino, for example.
  • an --OH group is replaced by a substituent which may, for example, be an inert organic group or halogen, notably fluorine.
  • substituents are hydrocarbyl and hydrocarbyl substituted by halogen or --OH; the hydrocarbyl moiety may contain an ether or ester linkage, for example.
  • the present invention provides novel peptidyl serine protease inhibitors in which the P2-P1 natural peptide linkage is replaced by another linking moiety other than an N-substituted P2-P1 natural peptide linkage.
  • the invention enables the provision of compounds having beneficial properties as inhibitors of serine proteases and favourable subsite interactions, and retaining geometry suitable for binding at the active site of the enzyme. It also enables the provision of compounds with different combinations of properties compared to prior art compounds, thereby providing the benefit of choice.
  • the invention provides compounds of the formula I:
  • X is H or an amino protecting group and is bonded to the amino group of the N-terminal amino acid
  • n is an integer of from 0 to 5;
  • n is 0 or 1, provided that if n and m are both 0 then X is a group of the formula R 10 (CH 2 ) e COO-- or R 10 (CH 2 ) e SO 2 -- wherein e is 0 to 3 and R 10 is a C 5 -C 12 aryl, arylalkyl or alkylaryl group optionally substituted by halogen or --OH;
  • ⁇ is --CO 2 --, --CH 2 O--, --NHCO--, --CHYCH 2 --, --CH ⁇ CH--, --CO(CH 2 ) p CO-- where p is 1, 2 or 3, --COCHY--, --CO 2 --CH 2 NH--, --CHY--NX--, --N(X)CH 2 N(X)CO--, --CH ⁇ C(CN)CO--, --CH(OH)--NH--, --CH(CN)--NH--, --CH(OH)--CH 2 -- or --NH---CHOH--, where X is H or an amino protecting group and Y is H or F;
  • aa 1 , aa 2 , aa 3 and aa 4 are each independently a residue of a natural or an unnatural amino acid or a group of the formula
  • a is 0 or 1
  • e 1;
  • b and d are independently 0 or an integer such that (b+d) is from 0 to 4;and (b+e) is from 1 to 4;
  • c is 0 or 1;
  • D is O or S
  • E is H, C 1-C 6 alkyl, or a saturated or unsaturated cyclic group which is a 5-6 membered ring, or an 8-14 membered fused ring system, which alkyl or cyclic group is optionally substituted by up to 3 groups independently selected from --R 13 , --R 1 OR 13 , --R 1 COR 13 , --R 1 CO 2 R 13 and --R 1 O 2 CR 13 , wherein R 1 is --(CH 2 ) f -- and R 13 is --(CH 2 ) g H or a moiety which has a total number of carbon and heteroatoms from 5 to 10 and which contains a ring system and optionally an alkyl and/or an alkylene group, wherein f and g are each independently from 0 to 10, provided that (f+g) does not exceed 10, and provided that there is only a single substituent if the substituent group is a said moiety containing a ring system, or E is C 1 -C 6 trialkylsilyl;
  • W 1 and W 2 together with the carbon atom to which they are bonded form a ring system W 1 and W 2 together form an alkenyl or aralkenyl group, or --HNC(W 1 )(W 2 )CO-- is the residue of an amino acid in which W 1 is H and W 2 is a group which together with the ⁇ -amino group forms a cyclic group which is a 4-6 membered ring or an 8-10 membered fused ring system optionally substituted by up to 3 groups independently selected from --R 13 , --R 1 OR 13 , --R 1 COR 13 , --R 1 CO 2 R 13 and --R 1 O 2 CR 13 , wherein R 1 and R 13 are as hereinbefore defined and any one or more hydrogen atoms bonded to a carbon atom is optionally replaced by halogen; and ##STR2## where: R 2 and R 3 are each independently selected from halogen, --OH, --OR 4 and --NR 4 R 5 , where R 4 and
  • R 2 and R 3 taken together represent a residue of a diol or a dithiol
  • R 7 and R 8 are each independently selected from the group consisting of R 2 , R 3 , R 4 and R 5 ;
  • R 9 is a group selected from the following: --H, --OR 4 , OR 5 ,
  • aa 2 is not a group of the formula --HNC(W 1 )(W 2 )CO-- wherein one of W 1 and W 2 is a group as defined in clause (i) above.
  • a third aspect of the invention resides in compounds of the formula II:
  • A is a group selected to have affinity for the specificity pocket of a serine protease, especially a trypsin-like protease
  • W is a moiety selected to have affinity for a binding subsite of a serine protease, especially a trypsin-like protease
  • ⁇ is a linker between W and A other than a natural peptide group or an N-substituted natural peptide group
  • Z is a C-terminal carboxy group or a replacement therefor.
  • natural amino acid means an L-amino acid (or a residue thereof) selected from the group consisting of
  • Trp tryptophan
  • unnatural amino acid is meant any ⁇ -amino acid (or residue thereof) other than the natural amino acids listed above.
  • Unnatural amino acids therefore include the D-isomers of the natural L-amino acids.
  • Examples of unnatural amino acids include for instance: D-Phe, norleucine, hydroxyproline, ⁇ -carboxyglutamic acid, pyroglutamic acid, and other amino acids having side chain protecting groups and which are capable of incorporation into the peptides of the invention,
  • analogue when used in reference to amino acid residues or other moieties denotes an alternative to another group without implying that analogous groups impart the same properties to a compound. To the contrary, biological properties of compounds can be significantly chanced by replacing a moiety with an analogue thereof.
  • Aa p phosphonic acid analogue of Aa
  • Chg cyclohexylglycine
  • NASO 2 napthylsulfonyl
  • Phg phenylglycine
  • p-TBDPS-O-Me p-tertbutyldiphenylsilyloxymethyl-phenylalanine
  • aryl as used herein includes aryl groups containing heteroatoms, i.e. heteroaryl groups.
  • alkyl includes cycloalkyl and alkyl containing cycloalkyl, where cycloalkyl is in particular cyclohexyl or cyclopentyl.
  • amino protecting group means any amino protecting group employable in peptide synthesis. Examples include: alkyl (especially methyl or other C 1 -C 6 alkyl), acetyl, benzoyl, BPoc, formyl, morpholinocarbonyl, trifluoroacetyl, methoxysuccinyl, aromatic urethane protecting groups such as benzyloxycarbonyl, aliphatic urethane protecting groups such as tertbutyloxycarbonyl or adamantyloxycarbonyl. Amino protecting groups are described in Gross and Meinhoffer, eds., The Peptides, Vol. 3, 3-88, and exemplified in D. W. Greene, "Protecting Groups in Organic Synthesis".
  • Preferred amino protecting groups include: R 10 (CH 2 ) c OCO-- or R 10 (CH 2 ) e SO 2 --, where R 10 is a C 5 -C 2 , preferably C 6 -C 10 , aryl, arylalkyl or alkylaryl group optionally substituted by halogen or --OH, especially phenyl, naphthyl or C 1 -C 4 alkylphenyl, and e is 0 to 3.
  • N-protecting groups are desirable in the compound structure during synthesis. These protecting groups are optionally removed or exchanged in the final structure.
  • Mtr (4-methoxy-2,3,6-trimethyl-benzenesulphonyl) or Pmc (2,2,5,7,8-pentamethylchroman-6-sulphate) may be used to protect Arg and Dtt (dithiothreitol) to protect Lys.
  • amino acid residues having acidic or hydroxy side chains may be suitably protected in the form of t-butyl, benzyl or other suitable esters or ethers, as is known in the art (e.g. Sheppard--"Solid Phase Peptide Synthesis, E. Atherton, R. C. Sheppard, IRL Press, Oxford, 1989).
  • salts include acid addition salts, e.g., salts of benzene sulphonic acid (BSA), hydrochloric acid (HCl) hydrobromic acid (HBr), acetic acid, trifluoroacetic acid (TFA), succinic acid, citric acid and other addition salt-forming acids known in the art.
  • BSA benzene sulphonic acid
  • HCl hydrochloric acid
  • HBr hydrobromic acid
  • acetic acid acetic acid
  • TFA trifluoroacetic acid
  • succinic acid citric acid and other addition salt-forming acids known in the art.
  • peptides (or, more precisely, peptide analogues) which are modified by, in particular, isosteric replacement of one or more remaining peptide bonds by --CO--CH 2 --, --CH(OH)--CH 2 or --CH 2 --NH-- linkages, or by N 4 .
  • the peptides may be in the free form or in a form protected at one or more remaining functional groups, e.g., amino, imino or amide (including peptide), nitro, carboxyl, hydroxyl, guanidino or nitrile. Examples of, and synthetic routes to, such further modifications of peptides are disclosed in for example EP-A-0118280 and corresponding U.S. Pat. Nos. 4,638,047 and 4,772,686, the disclosures of both of which references are incorporated herein by references, as well as in WO 92/07869.
  • the present invention has been shown to comprise peptides which exhibit good, and in many cases excellent, inhibitory properties with respect to a variety of serine proteases.
  • enzymes include trypsin-like enzymes such as thrombin.
  • Factor Xa and Factor VIIa and chymotrypsin-like enzymes such as elastase.
  • the moiety on the C-terminal side of the P1 residue is not critical to the invention. It is a moiety which interacts with the active site triad residues (Asp-His-Ser) of a serine protease.
  • the P1 residue is linked on its C-terminal side to a functional group which may be a carboxyl group (--COOH) or a derivative thereof, such as an ester, an amide or a ketone, for example, or even a nitrile group,
  • Z does not comprise a peptide linkage, or a replacement therefor ( ⁇ ), to a P1 1 amino acid residue.
  • the natural carboxy group is replaced by a heteroatom acid group, of which the preferred examples are boron or phosphorus acid groups, notably boronic acid residues [--B(OH) 2 ], phosphonic acid residues [--P(O)(OH) 2 ], phosphorous acid residues [--P(OH 2 )] or phosphinic acid residues [--P(O)(OH)(H)].
  • boron or phosphorus acid groups notably boronic acid residues [--B(OH) 2 ], phosphonic acid residues [--P(O)(OH) 2 ], phosphorous acid residues [--P(OH 2 )] or phosphinic acid residues [--P(O)(OH)(H)].
  • a less preferred heteroatom acid group is sulphonyl [--S(O) 2 OH].
  • heteroatom acid group there may be used a derivative thereof.
  • the invention is not primarily concerned with selection of derivatives of the carboxy or heteroatom acid groups: in principle, any derivative group may be used which does not prevent the inhibiting function of the compound.
  • Substituent groups include inert organic groups, generally containing a total number of carbon atoms and heteroatoms not exceeding 20. Representative inert groups are hydrocarbyl, optionally containing an ether or ester linkage and/or substituted by halogen or --OH.
  • the acid derivatives have the hydrogen of an --OH group replaced by a substituent group, which may be linked to the oxygen by a functional group, for example a carbonyl or amino group.
  • substituents are diol residues, as further described below.
  • an --OH group is replaced by an amino group, which may be mono- or di-substituted.
  • An alternative replacement functional group is thiol, especially substituted thiol.
  • an --OH group is replaced by an inert organic group (e.g. a hydrocarbyl group as described above) or by a halogen atom, especially fluorine.
  • One class of compounds has a C-terminal group (Z of formula I or II of the formula III:
  • Het is a heteroatom
  • s 0, 1 or 2;
  • t is the valency of Het, t-2s being an integer of at least 1, and each Y is independently hydrogen, halogen, hydroxy, substituted hydroxy, substituted thiol, amino or substituted amino, wherein two hydroxy groups, two thiol groups or an amino group are/is optionally substituted by a single divalent substituent.
  • Het is preferably boron or phosphorus, and most preferably boron.
  • each Y is independently F or other halogen, O ⁇ 1 or N ⁇ 1 ⁇ 2 , wherein ⁇ 1 and ⁇ 2 are independently selected from H, hydrocarbyl and hydrocarbylcarbonyl, the hydrocarbyl groups optionally being substituted by one or more moieties selected from halogen, --OH or alkoxy and/or containing an ether or ester linkage (--O-- or --COO--), which groups contain up to 20 carbon atoms, or wherein two Y groups taken together form the residue of a diol or a dithiol.
  • Particularly preferred C-terminal groups are of the formula ##STR3## wherein: R 2 and R 3 are each independently selected from halogen, --OH, --OR 4 and --NR 4 R 5 , where R 4 and R 5 are each independently a group of the formula R 6 (CO) u --, wherein u is 0 or 1, R 6 is H or an optionally halogenated alkyl, aryl or arylalkyl group containing up to (10-u) carbon atoms and optionally substituted by one or more groups selected from --OH, R 7 (CO) v O-- and R 7 (CO) v --, wherein v is 0 or 1.
  • R 7 is C 1 -C 6-v alkyl, or is an aryl, alkylaryl, arylalkyl or alkylarylalkyl group containing up to (10-v) carbon atoms.
  • R 2 and R 3 taken together represent a residue of a diol or a dithiol
  • R 7 and R 8 are each independently selected from the group consisting of R 2 , R 3 , R 4 and R 5 ;
  • R 9 is a group selected from the following: --H, --OR 4 , --OR 5 .
  • R 2 and R 3 are preferably --OR 4 in which R 4 is preferably a said optionally halogenated alkyl, aryl or arylalkyl group optionally substituted as aforesaid.
  • the diol or dithiol preferably comprises two or more --OH or, as the case may be, --SH groups connected by at least two connecting atoms.
  • the connecting atoms are preferably in an organic moiety containing up to 20 and, more preferably, up to 10 carbon atoms.
  • the organic moiety may be a hydrocarbyl group optionally containing between the members of one or two pairs of adjacent carbon atoms an N, S or O atom.
  • the organic moiety may be inertly substituted. Normally the substituted compounds are mono- or di- substituted, exemplary substituents being halogen especially --F, and --OH.
  • Preferred diol residues are of pinanediol, pinacol, perfluoropinacol, ethylene glycol, diethylene glycol, catechol, 1,2-cyclohexanediol, 1,2-cyclohexaneethanediol, 1,3-propanediol, 2,3-butanediol, 1,2-butanediol, 1,4-butanediol, 2,3-dimethylbutane-2-3-diol, glycerol, or diethanolamine or another amino dihydroxy alcohol.
  • pinanediol and especially pinacol are most preferred.
  • the most preferred compounds comprise a boronic acid residue substituted with a diol residue.
  • the C-terminal acid group may be bonded to an anion-binding exosite association moiety through an 18 ⁇ -42 ⁇ linker group.
  • the compounds of the invention are all characterised in that a natural peptide linkage (--NHCO--) is replaced by an alternative linker group.
  • the replaced peptide link is defined as the P2-P1 link in the first aspect of the invention and represented by ⁇ in formulae I and II. For convenience, the symbol ⁇ will hereafter be used.
  • ⁇ is a group which may be included in a compound of the invention without the inhibiting activity of the compound being lost.
  • Preferred ⁇ groups enhance the inhibitory activity of the compound. If ⁇ is long, there is a tendency for binding of the peptide inhibitor to the target enzyme to be weakened. Typically, therefore, ⁇ has a chain length of no more than 5 atoms, i.e. no more than 5 atoms separate the carbon atoms of the residues linked by ⁇ . More preferred ⁇ groups have a chain length of 2 or 3 atoms, a chain length of two atoms being most preferred.
  • ⁇ is preferably not isoelectronic with --NHCO-- .
  • One less preferred class of embodiments does not have ⁇ groups of the so-called isosteric (to --CONH--) type, such as --COCH 2 --, --CH(OH)--CH 2 --, --CH 2 --NX-- or --NHCO--, for example.
  • --COCH 2 -- and --CH(OH)--CH 2 -- are very acceptable in some compounds.
  • ⁇ groups include --CO 2 --, --CH 2 O--, --NHCO--, --CHYCH 2 --, --CH ⁇ CH--CO(CH 2 ) p CO-- where p is 1, 2 or 3, --COCHY--, --CO 2 --CH 2 NH--, --CHY--NX--, --N(X)CH 2 --N(X)CO--, --CH ⁇ C(CN)CO--, --CH(OH)--NH--, --CH(CN)--NH---, --CH(OH)--CH 2 or --NH---CHOH--, where X is H, an amino protecting group (e.g. CH 3 ) and Y is H or halogen (especially F).
  • exemplary Y-containing groups are --CH 2 CH 2 --, --COCHF-- and --CH 2 NX---.
  • the most preferred ⁇ groups are --CO 2 -- and --CH 2 O--.
  • the N-terminal group (X of Formula I) may be hydrogen (to form an --NH 2 group) or an amino protecting group.
  • the amino protecting group of the pharmaceutical compounds may be any pharmaceutically acceptable group, for example as described hereinbefore.
  • Alkyl groups e.g. C 1-C 6 alkyl such as methyl, for example, are suitable.
  • a preferred class of protecting groups are those of the formula R 10 (CH 2 ) e OCO-- and R 10 (CH 2 ) e SO 2 --, wherein e is 0, 1, 2 or 3 and R 10 is a C 5 -C 12 aryl, C 5 -C 12 arylalkyl or C 5 -C 12 alkylaryl group optionally substituted by halogen (e.g.
  • R 10 groups when m and n are 0, or when m is 0 and n is 1, are phenyl, naphthyl, C 1 -C 4 alkylphenyl or phenyl C 1 -C 4 alkyl.
  • e is 0.
  • N-terminal groups can enhance bioavailability of active compounds, although not necessarily effecting potency against the isolated target enzyme.
  • Typical groups of the active compounds include morpholin-N-alkyl or N-carbonyl derivatives, succinimidyl, alkyl or aryl-alkyl-sulphonyl, N-methylpiperazine or groups as known in the art, such as Rosenberg, et al. J.Med.Chem., 1993, 36, 449-459 or Hashimoto, N. et al. Pharm.Res., 1994, 11, 1443-1451, or Bernstein, P. R., et al. J.Med.Chem., 1994, 37, 3313-3326.
  • These groups can be introduced to the peptides by hydrogenation to remove urethane protecting groups used for synthesis to give the free amino terminus (see Example 2) and reacylation or acetylation with a derivative of the appropriate X group.
  • N-methyl groups can improve in-vivo activity as is known in the art, Hashimoto, N. et al Pharm.Res., 1994, 11, 1443-1451.
  • Peptide serine protease inhibitors comprise a sequence of amino acid residues and are commonly tripeptides.
  • the specific sequence is not critical to the invention.
  • the amino acids may be natural or unnatural, e.g. the D-isomer or racemate of a natural amino acid; they may be modified amino acids in which the ⁇ -H is replaced by a substituent, for example hydrophobic or hydrophilic groups containing up to about 20 or even more, e.g. 22, carbon atoms. More preferred substituents contain up to 15, or preferably up to 10, carbon atoms
  • a is 0 or 1
  • e 1;
  • b and d are independently 0 or an integer such that (b+d) is from 0 to 4 and (b-e) is from 1 to 4;
  • c is 0 or 1;
  • D is O or S
  • E is H, C 1 -C 6 alkyl, or a saturated or unsaturated cyclic group which normally contains up to 14 members and preferably is a 5-6 membered ring or an 8-14 membered fused ring system, which alkyl or cyclic group is optionally substituted by up to 3 groups (e.g.
  • R 1 group independently selected from --R 13 , --R 1 OR 13 , --R 1 COR 13 , --R 1 CO 2 R 13 , --R 1 O 2 CR 13 , nitro and cyano, wherein R 1 is --(CH 2 ) f and R 13 is --(CH 2 ) g H or a moiety which has a total number of carbon and heteroatoms from 5 to 10 and which contains a ring system (e.g.
  • f and g are each independently from 0 to 10, g preferably being at least 1 except that --OH is a preferred substituent, provided that (f+g) does not exceed 10, preferably does not exceed 6 and more preferably is 1, 2, 3 or 4, and provided that there is only a single substituent if the substituent is a said moiety containing a ring system, or E is C 1 -C 6 trialkylsilyl; and E 1 and E 2 are each independently a 5 or 6 membered ring;
  • a is 0. If a is 1, c is preferably 0. Preferably, (a+b+c+d) and (a+b+c+e) are no more than 4 and are more preferably 1, 2 or 3. (a+b+c+d) may be 0.
  • Exemplary groups for E, E 1 and E 2 include aromatic rings such as phenyl, naphthyl, pyridyl, quinolinyl and furanyl, for example; non-aromatic unsaturated rings, for example cyclohexenyl: saturated rings such as cyclohexyl, for example; and fused ring systems containing both aromatic and non-aromatic rings, for example fluorenyl.
  • a preferred class of E, E 1 and E 2 groups are aromatic rings, especially 6- membered aromatic rings.
  • E 1 and E 2 are preferably phenyl.
  • the phenyl or other aryl groups may be substituted by nitro or cyano, preferably at the 4-position.
  • E contains a substituent which is C 1 -C 6 alkyl, (C 1 -C 5 alkyl)carbonyl, carboxy C 1 -C 5 alkyl, aryl, especially 5-membered or preferably 6-membered aryl (e.g. phenyl or pyridyl), or arylalkyl (e.g. arylmethyl or arylethyl where aryl is preferably 6-membered).
  • aryl especially 5-membered or preferably 6-membered aryl (e.g. phenyl or pyridyl), or arylalkyl (e.g. arylmethyl or arylethyl where aryl is preferably 6-membered).
  • E contains a substituent which is OR 13 , wherein R 13 preferably is a 6-membered ring, which may be aromatic (e.g. phenyl) or non-aromatic (e.g. morpholine or piperazine) or is alkyl (e.g. methyl or ethyl) substituted by such a 6-membered ring.
  • R 13 preferably is a 6-membered ring, which may be aromatic (e.g. phenyl) or non-aromatic (e.g. morpholine or piperazine) or is alkyl (e.g. methyl or ethyl) substituted by such a 6-membered ring.
  • a particularly preferred class of moieties of formula V or VI are those in which E is a 6-membered aromatic ring substituted, preferably at the 2-position or 4-position, by --R 13 or --OR 13 .
  • a further preferred class of substituents of formula V or VI are of the formula C q H 2q T or ##STR5## wherein q is as defined above and T is hydrogen, halogen (e.g. F), --SiMe 3 , --R 13 , --COR 13 , CO 2 R 13 , --O 2 CR 13 or a moiety which has a total number of heteroatoms from 5 to 10 and which contains a ring system, especially an aryl group, and optionally an alkyl residue or an alkylene residue, or both.
  • Said moiety is preferably 5-membered or more preferably 6-membered aryl (e.g. phenyl or pyridyl) or arylalkyl (e.g.
  • T is at the 2-position of the phenyl group and is --R 13 , --COR 13 , --CO 2 R 13 or --O 2 CR 13 , and R 13 is C 1 -C 10 alkyl and more preferably C 1 -C 6 alkyl.
  • a class of residues which includes certain natural amino acid residues as well as many unnatural amino acid residues is of the formula
  • W 1 and W 2 may be the same or different and are selected from hydrogen and hydrogen replacement groups (i), (ii) and (iii) described above in relation to amino acid residues in which the ⁇ -hydrogen is replaced by a substituent; preferably, one of W 1 and W 2 is hydrogen.
  • W 1 and W 2 together with the carbon atom to which they arc bonded form a ring system, especially a hydrophobic ring system such as cycloalkyl (e.g. C 3 -C 7 cycloalkyl) or W 1 and W 2 together form an alkenyl or aralkenyl group, e.g.
  • the cyclic structure is preferably a 4-6 membered ring or an 8-10 membered fused ring system optionally substituted by up to 3 groups independently selected from --R 13 , --R 1 OR 13 , --R 1 COR 13 , --R 1 CO 2 R 13 and --R 1 O 2 CR 13 , wherein R 1 and R 13 are as hereinbefore defined.
  • exemplary substituents are C 1 -C 3 alkyl. Any one or more hydrogen atoms bonded to a carbon atom may optionally be replaced by halogen, especially F.
  • the cyclic structure may contain additional heteroatoms, for example sulphur, such as in a 5- or 6-member ring, for example.
  • a ring carbon atom may be a member of a carbonyl group, for example as part of an amide linkage in the cyclic structure, as in pyroglutamic acid, for example.
  • the cyclic structure preferably contains no heteroatom in addition to the ⁇ -amino nitrogen.
  • the ring fused to that containing the ⁇ -amino nitrogen is preferably aromatic and most preferably phenyl, as in D-Tiq.
  • WO 92/07869 and EP 0118280 disclose peptide inhibitors in which a P1 residue which is Arg or an Arg analogue is linked through a ⁇ linkage to P1 1 residue which is exemplified as Gly but may also be an amino acid residue with an optionally hydroxylated hydrocarbon side chain.
  • P1 1 residue which is exemplified as Gly but may also be an amino acid residue with an optionally hydroxylated hydrocarbon side chain.
  • aa 3 aa 2 is a sequence favoured by Kallikrein); in any event, in those compounds of this structure where aa 1 is glycine, aa 2 is not arginine, 3-(4 1 -amidinophenyl)-alanine or Gpa and normally is not any other amino acid whose side chain has a terminal amidino group, and more preferably is not any other arginine analogue as defined below.
  • the serine proteases are a widely studied family of enzymes, and a considerable body of knowledge exists as to amino acid sequences preferred by different enzymes.
  • the coagulation proteases are trypsin-like enzymes which in nature favour Arg, Lys or similar residues at P1.
  • An important factor for thrombin selectivity is the choice of P1 residue, for example by choosing methoxyalkyl as P1 residue.
  • Thrombin exhibits a preference for hydrophobic P2-P4 residues and, in the case of tripeptides favours D-configuration at P3.
  • Thrombin best accommodates inhibitors containing a P4 residue in which both the P3 and P4 residues are hydrophobic amino acids of L-configuration.
  • Particularly favoured (P4)P3P2 residues for some serine proteases are as follows:
  • Elastase is a chymotrypsin-like serine protease and favours phenylalanine and alanine and like (hydrophobic) amino acid residues at P1.
  • Plasmin and urokinase are trypsin-like.
  • amino acid residues may be replaced by analogue residues.
  • Preferred analogous residues of amino acids include those sharing the same polarity or charge.
  • Residues analogous to Lys or Arg and amongst the residues favoured by trypsin-like proteases at P1 are those with group (i) side chains and an ⁇ -hydrogen, that is, residues of the formula ##STR7## in which Q includes amino, amidino, imidazole, guanidino, N 3 or isothioureido.
  • Specific analogy residues to Lys and Arg include Gpa, amidinoPgl or amidinopiperidylglycine.
  • very acceptable P1 residues for the trypsin-like proteases are those with hydrophobic side chains, including Phe and its analogues.
  • Suitable hydrophobic side chains for the P1 residue include group (iii) side chains of Formula V. especially those in which a is 0, D is O or is absent and/or E is H, C 1 -C 6 alkyl, C 1 -C 6 trialkylsilyl or C 6 -C 10 aryl optionally substituted by up to three groups selected from C 1 -C 4 alkyl, halogen and C 1 -C 4 alkoxy, of which H is less preferred.
  • the Formula V groups contain a total number of carbon atoms and heteroatoms not exceeding 14, more preferably not exceeding 10 and most preferably not exceeding 8.
  • Particularly preferred hydrophobic P1 side chains are C 1 -C 8 , preferably C 1 -C 6 , alkyl (e.g. ethyl, isopropyl, pentyl), alkoxyalkyl containing from 2 to 6 carbon atoms (e.g. methoxypropyl) and moieties containing a 5-10 membered aryl or heteroaryl group and optionally a total number of alkyl and/or alkylene carbon atoms not exceeding 4, especially phenyl C 1 -C 4 alkyl (e.g. phenylmethyl).
  • Any of the aforesaid alkyl or alkylene groups may be substituted by one, or more than one, halo atom, e.g. fluoro or bromo; thus bromopropyl, especially 3-bromopropyl, or other bromoalkyl (usually substituted by Br at the terminal carbon) is a preferred P1 side chain.
  • Methoxyalkyl is a particularly preferred side chain.
  • the P1 side chain is C 1 -C 6 hydroxyalkyl, 3-methoxypropyl, 3-halopropyl and 3-hydroxypropyl and alkyl homologues thereof are particularly preferred.
  • W of formula II normally comprises a sequence of up to 9 amino acids, and more usually of up to 7 amino acids, wherein at least one amino acid has a hydrophobic side chain, e.g. Phe or a Phe analogue.
  • the P3 (aa 3 ) residue is desirably hydrophobic: the P2 residue (aa 2 ) is also preferably hydrophobic and more preferably is Pro or a ring homologue thereof.
  • Any P4 residue of a thrombin inhibitor is preferably also hydrophobic.
  • Residues analogous to Phe include those with group (iii) side chains and those of formula IX and those in which W 1 and W2 together form a hydrophobic ring system or an alkenyl or aralkenyl group.
  • p-halophenyl especially p-iodophenyl
  • a C 1 -C 6 group which is alkyl or alkyl substituted or interrupted by a carbonyl or carbonyloxy group (e.g. alkylcarbonyl or alkoxycarbonyl) or substituted by --R 14 or --OR 14 wherein R 14 is a 5- or 6-membered aromatic or non-aromatic ring or is C 1 -C 4 alkyl substituted by such a 6-membered ring; bipyridyl; furanyl; chromanyl; quinolinyl; thienyl; pyridyl; ⁇ - or ⁇ -naphthyl; thionaphthyl; indolyl; p-iodophenylalanyl; diphenyl-methyl; or fluorenyl; or are wholly or partially saturated groups corresponding to any of these (e.g.
  • any of the foregoing groups is optionally substituted by up to three groups selected from C 1 -C 3 alkyl, C 1 -C 3 alkoxy, R 13a CO-- wherein R 13a is H. CH 3 or C 2 H 5 , R 13a OR 1a -- or R 13a COR 1a --, wherein R 1a is --CH 2 --, --C 2 H 4 -- or --C 3 H 6 --.
  • L 1 and L 2 are each independently selected from the group consisting of CH 2 , CH 2 --CH 2 , O--CH 2 , S--CH 2 , and a bond.
  • V is H, or --NHV and one of Ar 1 --L 1 and Ar 2 --L 2 together form a group of the formula ##STR11##
  • L 1 or L 2 is a single bond, its attached Ar group be diphenylmethyl, fluorenyl or cyclohexyl.
  • Ar 2 L 2 is H.
  • Particularly preferred Phe analogues for the P3 residue are D-Phe substituted at the phenyl 2-position (i) by a C 1 -C 6 group which is alkyl or alkyl substituted or interrupted by a carbonyl or carbonyloxy group (e.g. is alkylcarbonyl or alkyloxycarbonyl) or (ii) by a 5 or 6 membered aryl group; D-Dpa; Dba: Pms; ⁇ - or ⁇ Nal; TMSal; Chg; Phg; D-Tiq or a para ether of D-Tyr.
  • An exemplary substituted phenylalanine residue is D-phenylalanine-2-carboxylic acid methyl ester.
  • Exemplary tyrosine-para-ethers are D-tyrosine-O-phenyl, D-tyrosine-O-ethyl-2-(N-morpholine) and D-tyrosine-O-ethyl-2-N(piperazine).
  • the most preferred Phe analogues are Dpa. Nal and Dba.
  • Other preferred Phe analogues for in particular the P3 residue have side chain c).
  • the compounds of the invention may take the form of monopeptides of the formula X- ⁇ -aa 1 , wherein X is R 10 (CH 2 ) e COO-- or R 10 (CH 2 ) e SO 2 -- and aa 1 is suitably a hydrophobic residue.
  • Particularly preferred R 10 groups are C 9 -C 10 fused ring systems containing a phenyl ring, especially naphthyl. Where R 10 is a fused ring system, e is preferably 0; if R 10 is a single ring, e may suitably be 1.
  • the residue of the acid function --COO-- or --SO 2 -- is preferably --SO 2 --.
  • Particularly preferred amino protecting group analogues for Phe are benzyloxycarbonyl (Cbz) and naphthylsulfonyl.
  • R 11 --CH 2 --, --CH 2 --CH 2 --, --S--CH 2 --, --S--C(CH 3 ) 2 -- or --CH 2 --CH 2 --CH 2 --.
  • Up to 3 C 1 -C 3 alkyl groups, e.g. methyl, may substitute 1 or more carbon atoms. Normally any substituent is on a --CH 2 -- group. Normally a --CH 2 -- group is substituted by no more than 1 alkyl group.
  • proline analogues are 2- and 3-thioproline and pipecolic acid.
  • Inhibitors of thrombin, and certain other inhibitors as indicated below, preferably have proline at their P2 position.
  • Kallikrein inhibitors preferably have proline at their P3 position. These proline residues may be replaced by proline analogues.
  • residues which are an analogue of Phe, Arg or Lys preferably have an ⁇ -hydrogen, but the hydrogen may be replaced by another group, e.g. a W moiety.
  • preferred classes of P1 residues of the inventive compounds are (i) Arg, Lys and their analogues, and (ii) hydrophobic residues.
  • Particularly favoured (P4)P3P2 sequences for thrombin and six other enzymes are listed above; preferred inhibitors for these seven enzymes include those in which the (P4)P3P2 residues are the favoured ones or analogues thereof
  • the most preferred inhibitors are not restricted to the favoured residues and their analogues, as will be revealed by a study of the following Table A which indicates the most preferred (P4)P3P2 residues for the seven enzymes.
  • inhibitors it is especially desirable for inhibitors to include both a preferred P1 residue for the target enzyme and preferred subsite binding peptide sequences (e.g. P3P2) for the enzyme.
  • P3P2 preferred subsite binding peptide sequences
  • tripeptide inhibitors are preferred, especially tripeptide boronates, and a particularly preferred sequence is PhePro- ⁇ -BoroMpg, especially inhibitors of the formula
  • the P1 Mpg residue may be replaced by Pgl.
  • Residues may be in either D- or L-configuration. D-configuration is preferred for the P3 residue of thrombin inhibitors.
  • the essential feature of the inventive compounds is their possession of a replacement bond ( ⁇ ) for a natural peptide bond as defined.
  • Other features of the compounds are not of the essence, provided that a compound inhibits its target enzyme.
  • the compounds of the invention may therefore be in the form of a pharmaceutically acceptable salt thereof and/or comprise one or more protectable functional groups (e.g. --OH or --NH 2 ) protected by a pharmaceutically acceptable protecting group.
  • Suitable salts include acid addition salts, as described above, and those of acid groups with Group I or Group II metal cations (e.g. Na + , K + , Mg 2+ , Ca 2+ ).
  • protecting groups of protectable functional groups there may be mentioned t-butyl and benzyl as protecting groups for --OH and --COOH functions.
  • the invention encompasses peptides in which one or more amide linkages other than the P2-P1 linkage are also replaced by an unnatural linker ⁇ , e.g. a preferred ⁇ group of this invention or, more preferably, a so-called isosteric group, e.g. --COCH 2 --, --CH(OH)--CH 2 -- or --CH 2 --NH 2 --.
  • an unnatural linker ⁇ e.g. a preferred ⁇ group of this invention or, more preferably, a so-called isosteric group, e.g. --COCH 2 --, --CH(OH)--CH 2 -- or --CH 2 --NH 2 --.
  • thrombin inhibitor molecule of a thrombin anion exosite association moiety.
  • thrombin anion binding exosite association moiety ABEAM
  • the ABEAM domain may comprise an) moiety which binds to the anion binding site of the target protease. Examples include amino acids 56-64 of hirudin, amino acids 1675-1686 of Factor V. amino acids 272-285 of platelet glycoprotein Ib, amino acids 415-428 of thrombomodulin, amino acids 245-259 of prothrombin Fragment 2 and amino acids 30 to 44 of fibrinogen A ⁇ chain.
  • the ABEAM component may be selected from any of the hirudin peptide analogues described by J. L. Krystenansky et al. "Development of MDL-28. 050. A small Stable Anththrombin Agent Based On A Functional Domain of the Leech Protein, Hirudin". Thromb, Haemostas., 63. pp. 208-14 (1990).
  • WO 91/02750 describes that the catalytic site-directed moiety of a thrombin inhibitor is linked to an ABEAM through a linker having a length of from 18 ⁇ to 42 ⁇ .
  • the linker which may be an amino acid sequence, is exemplified as bridging the C terminal of the catalytic site-directed moiety (CSDM) and the N-terminal of the ABEAM.
  • a representative ABEAM containing structure of the invention is:
  • LINKER may be 7-residue peptide.
  • the C-terminal boronic acid residue of the CSDM domain may be replaced by another heteroatom acid residue, e.g. a phosphonic acid residue.
  • the inhibitors compounds of the invention have affinity for one or more serine proteases.
  • the serine protease may be chymotrypsin-like or, more preferably, trypsin-like.
  • Exemplary enzymes are thrombin, kallikrein, elastase.
  • Factor Xa Factor Xa
  • Factor VIIa plasmin and urokinase.
  • the most preferred enzymes have affinity for thrombin.
  • Ki inhibition constant
  • a target enzyme 0.5 ⁇ M or less, preferably of 0.3 ⁇ M or less and most preferably of 0.1 ⁇ M or less.
  • Ki inhibition constant
  • a Ki of 0.05 ⁇ M or less is obtained, e.g. of about 0.035 to 0.04 ⁇ M (say, 0.039).
  • the Ki values herein refer to values determined at 37° C.
  • the inhibiting compounds prefferably be selective towards one enzyme, e.g. to have a Ki for the selected enzyme of 0.1 ⁇ M or less (e.g. of between about 0.035to 0.09 ⁇ M), and a Ki towards other serine proteases exceeding 0.1 ⁇ M and more preferably exceeding 0.2 ⁇ M, e.g. 0.25 ⁇ M or more.
  • the Ki towards non-selected enzymes may exceed 0.5 ⁇ M or 1 ⁇ M.
  • the ratio of Ki for non selected enzymes is preferably at least 2 and more preferably at least 3.
  • the Ki ratio may be at least 5.
  • novel peptides of the present invention can be prepared by using, for example, generally known peptide synthesis methods. It is convenient in many instances to premake as intermediates the binding subsite affinity moiety [X-(aa 4 ) m -(aa 3 ) n -(aa 2 ) of Formula I ]and the specificity pocket affinity moiety with its attached C-terminal group [(aa 1 )-Z of Formula I ].
  • the two intermediates contain suitable functional groups to react together to form the target non-natural amide bond [ ⁇ of Formula I] and are caused or allowed to react together to form the compound (or a precursor thereof to undergo one or more further functional group transformations).
  • the invention includes intermediates of the formula X-(aa 4 ) m -(aa 3 ) n -(aa 2 )-G 1 or W-G 1 and G 2 -(aa 1 )-Z or G 2 -A-Z, wherein G 1 and G 2 are groups which may be reacted together to form a linking group other than a natural amide bond, optionally after "working up” (e.g. hydrogenation) of the direct product.
  • G 1 is not --COOH (and sometimes is not an ester or other reactive derivative thereof) and G 2 is H 2 N--.
  • G 1 and G 2 groups are as follows:
  • Lg is a leaving group and M + is an alkali metal ion or another cation.
  • Species which contain a free hydroxyl group (OH), and G 2 is a leaving group, especially halogen, e.g. Cl, Br and the base DBU or an organolithium (e.g. Butyl lithium) are used.
  • Species which has an aldehyde (CHO) G 1 group and G 2 is an amino group, and the reagent sodium cyanoborohydride are used.
  • Ketomethylene bonds can be prepared by reaction of a unit X-(aa 4 ) m -(aa 3 ) n -(aa 2 )-carbonyldimmidazole and the lithium salt of tert-butyl acetate to give a beta-diketone X-(aa 4 ) m (aa 3 ) n (aa 2 )--COCH 2 COOtBU, and alkylation with NaH and a halomethylketone (Hoffman, R. V. and Kim, H. O., Tet.Lett.,1992, 33, 3597-3582) or ⁇ -haloboronate (e.g. Hal--CHRBO 2 Pin) or ⁇ -halophosphonate and subsequent hydrolysis.
  • ⁇ -haloboronate e.g. Hal--CHRBO 2 Pin
  • ⁇ (CH ⁇ CH) could be prepared by the methods described by Ibuka, T., Yoshizawa. H., Habashita, H., Fuji, N., Chounan, Y., Tanaka, M., and Yamamoto, Y., Tet. Lett., 1992, 33, 3783-3786 or Ibuka, T., Habashita, H., Otaka, A., Fuji, N., Oguchi. Y., Uyehara, T. and Yamamoto, Y., J.Org.Chem., 1991, 56, 4370-4382.
  • P2-P1 peptide bond replacements may be made as known in the art, such as Marraud, M., Dupont, V., Grand, V., Zerkout, S., Lecoq, A., Boussard, G., Vidal, J., Collet, A., and Aubry, A. "Modifications of the Amide Bond and Conformational Constraints in Pseudoamide Analogues",
  • the reaction is preferably carried out in a dry, aprotic, polar solvent for example tetrahydrofuran, at a temperature between about -79° C. and room temperature (typically, 20° C.).
  • a dry, aprotic, polar solvent for example tetrahydrofuran
  • the intermediates may be obtained by the methods disclosed herein or alternatively by general methods as described in Matteson et al, Organometallics, 3, 1284-8 (1984). or as in Elgendy et al, Tet.Lett, 1992, 33, 4209-4212 or Tetrahedron 1994, 50, 3803-3812 or Rangaishenvi et al, J.Org.Chem 1991, 56, 3286-3294, or in EP-A-0599633.
  • Suitable replaceable protecting groups may be used, for example as outlined for instance in Greene, T. W. and Wuts. P. G. M., "Protective Groups in Organic Chemistry", Wiley-Interscience, 1991.
  • the starting amino acid(s) for the preparation of the protected peptide of intermediate may be prepared by standard, well-known methods such as those described for example in Angew. Chem. 93, 793 (1981), J.Am Chem. Soc., 109, 6881 (1987) and J Jones, "The Chemical Synthesis of Peptides”. Oxford Science Publications, No. 23, Clarendon Press, Oxford 1992, or may be obtained from a variety of well known commercial sources
  • novel peptides according to the present invention are useful as inhibitors or substrates of various enzymes, particularly trypsin-like proteases, and may be used in vitro or in vivo for diagnostic and mechanistic studies of these enzymes. More generally, the novel peptides may be useful for research or synthetic purposes. Furthermore, because of their inhibitory action, the inhibitors are useful in the prevention or treatment of diseases caused by an excess of an enzyme in a regulatory system particularly a mammalian system, e.g. the human or animal body, for example control of the coagulation or fibrinolysis system.
  • the pharmaceutically useful compounds have a pharmaceutically acceptable group as any N-terminal substituent (X).
  • the compounds of the invention which are thrombin, kallikrein, factor Xa, or factor VIIa inhibitors have anti-thrombogenic properties and may be employed when an anti-thrombogenic agent is needed. Generally, these compounds may be administered orally or parenterally to a host in an effective amount to obtain an anti-thrombogenic effect. In the case of larger mammals such as humans, the compounds may be administered alone or in combination with one or more pharmaceutical carriers or diluents at a dose of from 0.02 to 10 mg/Kg of body weight and preferably 1-100 mg/Kg, to obtain the anti-thrombogenic effect, and may be given as a single dose or in divided doses or as a sustained release formulation. When an extracorporeal blood loop is to be established for a patient, 0.1-10 mg/Kg may be administered intravenously. For use with whole blood, from 1-100 mg per litre may be provided to prevent coagulation.
  • compositions for human or veterinary use are well known and include sugars, starches and water, and may be used to make acceptable formulations of pharmaceutical compositions (human or veterinary) containing one or more of the subject peptides in the required pharmaceutically appropriate or effective amount or concentration.
  • Formulations of the compounds include tablets, capsules, injectable solutions and the like.
  • the compounds of the invention may also be added to blood for the purpose of preventing coagulation of the blood in blood collecting or distribution containers, tubing or implantable apparatus which comes in contact with blood.
  • Advantages enabled by the invention include oral activity, rapid onset of activity and low toxicity.
  • these compounds may have special utility in the treatment of individuals who are hypersensitive to compounds such as heparin or other known inhibitors of thrombin or other serine proteases.
  • amino acid residues are of L-configuration unless otherwise stated.
  • the filtrate was concentrated under reduced pressure on a rotary evaporator, to afford a thick, viscous residue.
  • Preliminary examination by 1 H N.M.R. showed the required crude product.
  • the crude sample was dissolved in a small amount of MeOH, applied to the sephadex LH20 column, and then eluted with a pump using the same solvents.
  • the elution profile was followed with the aid of a U.V. lamp (226 nM) and recorder.
  • the void volume, fraction 1-6. and a further bulk volume were collected. From the shape of the chromatogram, it was deemed that fractions 1-6 would be the most likely fractions in which the tripeptide may be found.
  • the fractions were concentrated individually to afford clear slightly coloured viscous residues.
  • the organic phase was dried (by stirring over MgSO 4 ), filtered and concentrated under reduced pressure to give the crude product (1.018 g).
  • the crude product (1.018 g) was afforded as a thick semi-solid residue and was purified by gel filtration through Sephadex LH20. The fractions were pooled appropriately.
  • the purified yield was actually 41% based upon the yield of the 1-bromopropylboronate pinanediol derivative.
  • F4 was found to be the required product Cbz-D-Phe-Pro- ⁇ (CH 2 O)-BoroPhe pinanediol ester (rmm 657) and showed MS, 680 (M+Na), and a peak at 15 min on Rp HPLC (gradient 50-99% over 25 min, vydac 4.4 ⁇ 250 mm column).
  • the tertiary butyl ester (1.351 g, 4.21 mmol) was dissolved in 95% TFA (50ml) and stirred for 1 h under argon at room temperature The solvent was then pumped off under reduced pressure, to afford a white powder.
  • the product was authenticated by NMR, CHN, and FAB-Ms (rmm 265, yield 1.12 g, 100%).
  • the target compound was synthesised following the method of Example 4 using chloromethylbisoxophospholane(-)pinanediol (5.5 ⁇ 10 -4 mol) and Cbz-D-DpaPro (5.5 ⁇ 10 -4 mol). The reaction gave, after chromatography on Sephadex, the required product in 60% yield. ##STR21##
  • Examples 15-18 describe the preparation of O,O-Dialkyldipeptidyl carboxyphosphonates.
  • the preparation of the O,O-dialkyl- ⁇ -hydroxybenzyl phosphonates is described in Example 19.
  • Table 1 13 C N.M.R. characterisation data for various compounds, including examples of those according to the present invention.
  • Table 2 1 H N.M.R. characterisation data for various compounds, including examples of those according to the present invention.
  • Table 6 comparative Ki data for compounds of the invention and prior art compounds.
  • a volume of 150 ⁇ l of citrated normal human plasma and 20 ⁇ l of buffer or sample were warmed at 37° C. for 1 min. Coagulation was started by adding 150 ⁇ l of freshly prepared bovine thrombin (5NIHu/ml saline) and the coagulation time was recorded on a coagulometer.
  • bovine thrombin (5NIHu/ml saline)
  • a phosphate buffer, pH7.8, containing 0.1% bovine serum albumin and 0.02% sodium azide was used.
  • the samples were dissolved in DMSO and diluted with the buffer. When no inhibitor was used DMSO was added to the buffer to the same concentration as that used in the samples.
  • the inhibitor concentrations were plotted against the thrombin times in a semilogarithmic graph from which the inhibitor concentration that caused a doubling (40 sec) of the thrombin time was determined.
  • the inhibition of human ⁇ -thrombin was determined by the inhibition of the enzyme catalysed hydrolysis of three different concentrations of the chromogenic substrate S-2238.
  • the buffer used contained 0.1M sodium phosphate, 0.2M NaCl, 0.5% PEG and 0.02% sodium azide, adjusted to pH 7.5 with orthophosphoric acid.
  • the samples consist of the compound dissolved in DMSO.

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US08/894,120 1995-02-16 1996-02-15 Serine protease inhibitors Expired - Fee Related US6127340A (en)

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GB9502985 1995-02-16
GBGB9502985.6A GB9502985D0 (en) 1995-02-16 1995-02-16 Enzyme inhibitors
PCT/GB1996/000352 WO1996025427A1 (en) 1995-02-16 1996-02-15 Serine protease inhibitors

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US20040013575A1 (en) * 2002-05-13 2004-01-22 Becton, Dickinson And Company Protease inhibitor sample collection system
US20040268423A1 (en) * 2000-11-03 2004-12-30 Yadong Huang Methods of treating disorders related to apoE
US20050124965A1 (en) * 2003-12-08 2005-06-09 Becton, Dickinson And Company Phosphatase inhibitor sample collection system
US20050288253A1 (en) * 2002-09-09 2005-12-29 Trigen Limited Boronic acid salts
US20060212020A1 (en) * 2002-10-10 2006-09-21 Lynne Rainen Sample collection system with caspase inhibitor
US20110114835A1 (en) * 2008-06-05 2011-05-19 Micromass Uk Limited Method Of Charge Reduction Of Electron Transfer Dissociation Product Ions
US20130146762A1 (en) * 2007-11-23 2013-06-13 Micromass Uk Limited Mass Spectrometer

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AU2003263343A1 (en) 2002-09-09 2004-03-29 Trigen Limited Boronic acid salts ant their use in the treatment of thromobis
GB0405267D0 (en) * 2004-03-09 2004-04-21 Trigen Ltd Compounds

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WO1992007869A1 (en) * 1990-11-06 1992-05-14 Thrombosis Research Institute Inhibitors and substrates of thrombin
WO1994020526A1 (en) * 1993-03-03 1994-09-15 Sandoz Ltd. Peptide boronic acid derivatives having protease inhibiting activity
WO1995025427A1 (en) * 1994-03-18 1995-09-28 Nehls, Reinhard Ear tag for marking animals, and a pin which is suitable for use in an ear tag

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GB8305985D0 (en) * 1983-03-04 1983-04-07 Szelke M Enzyme inhibition

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
WO1992007869A1 (en) * 1990-11-06 1992-05-14 Thrombosis Research Institute Inhibitors and substrates of thrombin
WO1994020526A1 (en) * 1993-03-03 1994-09-15 Sandoz Ltd. Peptide boronic acid derivatives having protease inhibiting activity
WO1995025427A1 (en) * 1994-03-18 1995-09-28 Nehls, Reinhard Ear tag for marking animals, and a pin which is suitable for use in an ear tag

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7700304B2 (en) 2000-11-03 2010-04-20 The J. David Gladstone Institutes Methods of screening disorders related to apoE
US20040268423A1 (en) * 2000-11-03 2004-12-30 Yadong Huang Methods of treating disorders related to apoE
AU2002239766B9 (en) * 2000-11-03 2007-04-05 The J. David Gladstone Institutes Methods of treating disorders related to apoE
AU2002239766B2 (en) * 2000-11-03 2006-11-09 The J. David Gladstone Institutes Methods of treating disorders related to apoE
US20040013575A1 (en) * 2002-05-13 2004-01-22 Becton, Dickinson And Company Protease inhibitor sample collection system
US7309468B2 (en) 2002-05-13 2007-12-18 Becton, Dickinson And Company Protease inhibitor sample collection system
US7645425B2 (en) 2002-05-13 2010-01-12 Becton, Dickinson And Company Protease inhibitor sample collection system
US20080241001A1 (en) * 2002-05-13 2008-10-02 Becton, Dickinson And Company Protease Inhibitor Sample Collection System
US20050288253A1 (en) * 2002-09-09 2005-12-29 Trigen Limited Boronic acid salts
US20060212020A1 (en) * 2002-10-10 2006-09-21 Lynne Rainen Sample collection system with caspase inhibitor
US20050124965A1 (en) * 2003-12-08 2005-06-09 Becton, Dickinson And Company Phosphatase inhibitor sample collection system
US20130146762A1 (en) * 2007-11-23 2013-06-13 Micromass Uk Limited Mass Spectrometer
US9070540B2 (en) * 2007-11-23 2015-06-30 Micromass Uk Limited Mass spectrometer
US20110114835A1 (en) * 2008-06-05 2011-05-19 Micromass Uk Limited Method Of Charge Reduction Of Electron Transfer Dissociation Product Ions
US20140110576A1 (en) * 2008-06-05 2014-04-24 Micromass Uk Limited Method of Charge Reduction of Electron Transfer Dissociation Product Ions
US8624179B2 (en) * 2008-06-05 2014-01-07 Micromass Uk Limited Method of charge reduction of electron transfer dissociation product ions
US9070539B2 (en) * 2008-06-05 2015-06-30 Micromass Uk Limited Method of charge reduction of electron transfer dissociation product ions
US20150294846A1 (en) * 2008-06-05 2015-10-15 Micromass Uk Limited Method of Charge Reduction of Electron Transfer Dissociation Product Ions
US9761425B2 (en) * 2008-06-05 2017-09-12 Micromass Uk Limited Method of charge reduction of electron transfer dissociation product ions
US10490394B2 (en) * 2008-06-05 2019-11-26 Micromass Uk Limited Method of charge reduction of electron transfer dissociation product ions

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NZ301335A (en) 1998-07-28
EP0809650A1 (en) 1997-12-03
GB9603217D0 (en) 1996-04-17
AU707059B2 (en) 1999-07-01
ZA961225B (en) 1996-08-23
CA2212830A1 (en) 1996-08-22
WO1996025427A1 (en) 1996-08-22
GB9502985D0 (en) 1995-04-05
JPH11500433A (ja) 1999-01-12
GB2299583B (en) 1997-08-13
GB2299583A (en) 1996-10-09
AU4672996A (en) 1996-09-04

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